Ceramides (Cer) are comprised of a sphingoid base and amide-linked fatty acid, and are the backbones of complex sphingolipids as well as modulators of vital cellular processes. In recent years, it has become evident that mammalian tissues contain many different subcategories of Cer, and that the enzymes that form these important compounds are highly selective with respect to the fatty acyl-CoA substrate, but their selectivities for the sphingoid base have not yet been fully defined. Therefore, the overall objective of this grant is to provide a more fundamental and complete understanding of Cer synthases (CerS), their roles in regulating sphingoid base and Cer metabolism, and functions of novel metabolites. A major goal of the research in this grant is to elucidate the pathways for the biosynthesis and turnover of two recently discovered 1-deoxy- and 1-desoxymethyl-sphingoid bases as the backbones, and some of their biological functions (Aim 1). These compounds are made when serine palmitoyltransferase utilizes alanine or glycine instead of serine, and at least one known disease-human sensory neuropathy type 1, HSN1--has been found to result from elevated production of 1-deoxysphinganine (present mainly as the downstream metabolite 1- deoxydihydroCer). Preliminary studies for this proposal have established that production of these alternative categories of sphingolipids is far more common than has been previously appreciated, and the factors that influence their biosynthesis will be identified. Characterization of the CerS will also establish which are responsible for production of particular Cer subspecies, structural features of the enzymes that account for this selectivity, and determine how their activity is influenced by formation of homo- and hetero- dimers (Aim 2). These studies will utilize lipidomic mass spectrometry for the sphingolipid analysis because this technology provides information about not only individual molecular subspecies but also for other branches and metabolites. Thus, Aim 3 of the proposal will evaluate cross-talk among the different branches and metabolites and provide a integrative explanation for why distinctive subspecies distributions are found in plasma and tissues. Since many of the subspecies of sphingoid bases and Cer have been implicated in inherited and acquired disease, these studies will provide the underlying map of Cer metabolism that will help these processes be understood, and assist in developing more rational strategies for intervention.